Search

Vibrio vulnificus is a zoonotic pathogen that can cause death by septicaemia in farmed fish (mainly eels) and humans. The zoonotic strains that have been isolated from diseased eels and humans after eel handling belong to clade E (or serovar E (SerE)), a clonal complex within the pathovar (pv.) piscis. The aim of this study was to evaluate the accuracy of MALDI-TOF mass spectrometry (MS) in the identification of SerE, using the other two main pv. piscis-serovars (SerA and SerI) from eels as controls. MALDI-TOF data were compared with known serologic and genetic data of five pv. piscis isolates or strains, and with the non pv. piscis reference strain. Based on multiple spectra analysis, we found serovar-specific peaks that were of ~3098 Da and ~ 4045 Da for SerE, of ~3085 Da and ~ 4037 Da for SerA, and of ~3085 Da and ~ 4044 Da for SerI. Therefore, our results demonstrate that MALDI-TOF can be used to identify SerE and could also help in the identification of the other serovars of the species. This means that zoonosis due to V. vulnificus could be prevented by using MALDI-TOF, as action can be taken immediately after the isolation of a possible zoonotic V. vulnificus strain.

Vibrio vulnificus is a zoonotic pathogen that can cause death by septicaemia in farmed fish (mainly eels) and humans. The zoonotic strains that have been isolated from diseased eels and humans after eel handling belong to clade E (or serovar E (SerE)), a clonal complex within the pathovar (pv.) piscis. The aim of this study was to evaluate the accuracy of MALDI-TOF mass spectrometry (MS) in the identification of SerE, using the other two main pv. piscis-serovars (SerA and SerI) from eels as controls. MALDI-TOF data were compared with known serologic and genetic data of five pv. piscis isolates or strains, and with the non pv. piscis reference strain. Based on multiple spectra analysis, we found serovar-specific peaks that were of ~3098 Da and ~ 4045 Da for SerE, of ~3085 Da and ~ 4037 Da for SerA, and of ~3085 Da and ~ 4044 Da for SerI. Therefore, our results demonstrate that MALDI-TOF can be used to identify SerE and could also help in the identification of the other serovars of the species. This means that zoonosis due to V. vulnificus could be prevented by using MALDI-TOF, as action can be taken immediately after the isolation of a possible zoonotic V. vulnificus strain.

Despite vaccination, outbreaks of vibriosis still occur in sea-reared rainbow trout in Denmark. Vibriosis outbreaks are caused mainly by V. anguillarum serotypes O1 and O2a, and bacterins of both serotypes are included in the commonly used vaccine against this disease in Danish aquaculture. However, while the strains belonging to serotype O1 are genetically similar, the strains belonging to serotype O2a are highly diverse. This work aimed first at examining how the antibody response and protection induced by bacterin-based vaccines were affected by the antigenic variability within V. anguillarum serotype O2a strains. Following vaccination of rainbow trout with either a commercial or an experimental vaccine, specific antibody reactivity in serum from vaccinated fish was examined by ELISA against 23 strains of V. anguillarum serotype O2a (VaO2a). The strains were divided into 4 distinct subgroups according to the observed detection pattern. Seven strains were strongly recognized only by sera from fish vaccinated with the experimental vaccine (EV-I antisera), while 13 other strains were primarily recognized by sera from fish vaccinated with the commercial vaccine (CV antisera). Two strains were recognized by both EV-I and CV antisera, but with intermediate reactivity, while one strain was not recognized at all. A partly similar recognition pattern was observed when purified lipopolysaccharide (LPS) was used as antigen in the examination of antibody reactivity in Western blotting. The level of protection was highly dependent on both the vaccine and the strain used for challenge and showed no consistent correlation with antibody reactivity. Secondly, we attempted to use a bacterin vaccine based on one of the V. anguillarum O2a strains intermediately recognized by both EV-I and CV antisera to investigate whether that could potentially provide protection across strain variability. The immunized fish did mount a cross-reactive antibody re

Background: Prebiotics are known to have a positive impact on fish health and growth rate, and beta-glucans are among the most used prebiotics on the market. In this study, rainbow trout (Oncorhynchus mykiss) were treated with a beta-1,3;1,6-glucan dietary supplement (at a dose of 0 g, 1 g, 10 g, and 50 g beta-glucan per kg of feed). After 6 weeks, the effect of the beta-glucan was evaluated by determining the changes in the microbiota and the blood serum metabolites in the fish. The impact of beta-glucan on the immune system was evaluated through a challenge experiment with the bacterial fish pathogen Yersinia rucked.Results: The microbiota showed a significant change in terms of composition following beta-glucan treatment, notably an increase in the relative abundance of members of the genus Aurantimicrobium, associated with a decreased abundance of the genera Carnobacterium and Deefgea. Furthermore, analysis of more than 200 metabolites revealed that the relative levels of 53 metabolites, in particular compounds related to phosphatidylcholines, were up- or downregulated in response to the dietary supplementation, this included the amino acid alanine that was significantly upregulated in the fish that had received the highest dose of beta-glucan. Meanwhile, no strong effect could be detected on the resistance of the fish to the bacterial infection.Conclusions: The present study illustrates the ability of beta-glucans to modify the gut microbiota of fish, resulting in alteration of the metabolome and affecting fish health through the lipidome of rainbow trout.

Background: Although Flavobacterium psychrophilum is recognized as the causative pathogen of rainbow trout fry syndrome (RTFS), often resulting in high fry mortality, it is also responsible for bacterial cold water disease (BCWD) in large and older rainbow trout (Oncorhynchus mykiss). These older fish do not experience high mortality, but sustain, through the shedding of bacteria, a constant infection pressure at farm level, which exposes fry to an unnecessary infection risk. We have produced and assessed the immunogenicity of an experimental injection BCWD vaccine, which may be used to decrease the shedding of bacteria from older fish. Methods: A total of 800 fish were i.p.-injected: 200 fish received the bacterin with adjuvant, 200 fish received the bacterin alone, 200 fish received adjuvant alone and 200 fish were injected with physiological saline. Blood samples were taken at day 0 and at three different time points (4, 8 and 14 weeks) post-vaccination. Plasma antibody levels were measured by ELISA for reactivity against both the homologous F. psychrophilum vaccine strain (serotype Fd) and heterologous strains (serotype Th). Results: Significantly elevated antibody titers were found against all serotypes in vaccinated fish. Welfare parameters associated with the vaccination process were evaluated by analyzing trout plasma samples for six different biochemical parameters, but no adverse effects associated with injection were indicated. Conclusions: The study suggests that an injection vaccine containing formalin-inactivated whole cells of F. psychrophilum (serotype Fd), adjuvanted with FIA, may also induce protection against heterologous strains. We advocate for, as the next step, the performance of field trials evaluating if the vaccination of older rainbow trout will (1) reduce the infection pressure in farms, (2) elevate the general health level in all groups and (3) minimize F. psychrophilum infection in fry at farm level. This may reduce the need for the

Flavobacteria are among the most important pathogens in freshwater salmonid aquaculture worldwide. Due to concerns regarding development of antibiotic resistance, phage therapy has been proposed as a solution to decrease pathogen load. However, application of phages is challenged by the development of phage resistance, and knowledge of the mechanisms and implications of phage resistance is therefore required. To study this, 27 phage-resistant isolates of F. psychrophilum were genome sequenced and characterized to identify genetic modifications and evaluate changes in phenotypic traits, including virulence against rainbow trout. Phage-resistant isolates showed reduction or loss of gliding motility, proteolytic activity, and adhesion to surfaces, and most isolates were completely non-virulent against rainbow trout fry. Genomic analysis revealed that most phage-resistant isolates had mutations in genes associated with gliding motility and virulence. Reversal of these mutations in a sub-set of isolates led to regained motility, proteolytic activity, virulence and phage susceptibility. Although costly, the fast generation of phage resistance driven by single, reversible mutations likely represents a flexible and efficient phage defence mechanism in F. psychrophilum. The results further suggest that phage administration in aquaculture systems to prevent F. psychrophilum outbreaks selects for non-virulent phage-resistant phenotypes.

Background: Although Flavobacterium psychrophilum is recognized as the causative pathogen of rainbow trout fry syndrome (RTFS), often resulting in high fry mortality, it is also responsible for bacterial cold water disease (BCWD) in large and older rainbow trout (Oncorhynchus mykiss). These older fish do not experience high mortality, but sustain, through the shedding of bacteria, a constant infection pressure at farm level, which exposes fry to an unnecessary infection risk. We have produced and assessed the immunogenicity of an experimental injection BCWD vaccine, which may be used to decrease the shedding of bacteria from older fish. Methods: A total of 800 fish were i.p.-injected: 200 fish received the bacterin with adjuvant, 200 fish received the bacterin alone, 200 fish received adjuvant alone and 200 fish were injected with physiological saline. Blood samples were taken at day 0 and at three different time points (4, 8 and 14 weeks) post-vaccination. Plasma antibody levels were measured by ELISA for reactivity against both the homologous F. psychrophilum vaccine strain (serotype Fd) and heterologous strains (serotype Th). Results: Significantly elevated antibody titers were found against all serotypes in vaccinated fish. Welfare parameters associated with the vaccination process were evaluated by analyzing trout plasma samples for six different biochemical parameters, but no adverse effects associated with injection were indicated. Conclusions: The study suggests that an injection vaccine containing formalin-inactivated whole cells of F. psychrophilum (serotype Fd), adjuvanted with FIA, may also induce protection against heterologous strains. We advocate for, as the next step, the performance of field trials evaluating if the vaccination of older rainbow trout will (1) reduce the infection pressure in farms, (2) elevate the general health level in all groups and (3) minimize F. psychrophilum infection in fry at farm level. T

Flavobacteria are among the most important pathogens in freshwater salmonid aquaculture worldwide. Due to concerns regarding development of antibiotic resistance, phage therapy has been proposed as a solution to decrease pathogen load. However, application of phages is challenged by the development of phage resistance, and knowledge of the mechanisms and implications of phage resistance is therefore required. To study this, twenty-seven phage-resistant isolates of F. psychrophilum were genome sequenced and characterized to identify genetic modifications and evaluate changes in phenotypic traits, including virulence against rainbow trout. Phage-resistant isolates showed reduction or loss of gliding motility, proteolytic activity, and adhesion to surfaces, and most isolates were completely non-virulent against rainbow trout fry. Genomic analysis revealed that most phage resistant isolates had mutations in genes associated with gliding motility and virulence. Reversal of these mutations in a sub-set of isolates led to regained motility, proteolytic activity, virulence, and phage susceptibility. Although costly, the fast generation of phage resistance driven by single, reversible mutations likely represents a flexible and efficient phage defence mechanism in F. psychrophilum. The results further suggest that phage administration in aquaculture systems to prevent F. psychrophilum outbreaks selects for non-virulent phage resistant phenotypes.

The fish pathogen Flavobacterium psychrophilum is currently one of the main pathogenic bacteria hampering the productivity of salmonid farming worldwide. Although putative virulence determinants have been identified, the genetic basis for variation in virulence of F. psychrophilum is not fully understood. In this study, we analyzed whole-genome sequences of a collection of 25 F. psychrophilum isolates from Baltic Sea countries and compared genomic information with a previous determination of their virulence in juvenile rainbow trout. The results revealed a conserved population of F. psychrophilum that were consistently present across the Baltic Sea countries, with no clear association between genomic repertoire, phylogenomic, or gene distribution and virulence traits. However, analysis of the entire genome of four F. psychrophilum isolates by hybrid assembly pro-vided an unprecedented resolution for discriminating even highly related isolates. The results showed that isolates with different virulence phenotypes harbored genetic variances on a number of consecutive leucine-rich repeat (LRR) proteins, repetitive motifs in gliding motility-associated protein, and the insertion of transposable elements into intergenic and genic regions. Thus, these findings provide novel insights into the genetic variation of these elements and their putative role in the modulation of F. psychrophilum virulence.

The microbial community surrounding fish eyed eggs can harbor pathogenic bacteria. In this study we focused on rainbow trout (Oncorhynchus mykiss) eyed eggs and the potential of bacteriophages against the pathogenic bacteria Flavobacterium psychrophilum and F. columnare. An infection bath method was first established, and the effects of singular phages on fish eggs was assessed (survival of eyed eggs, interaction of phages with eyed eggs). Subsequently, bacteria-challenged eyed eggs were exposed to phages to evaluate their effects in controlling the bacterial population. Culture-based methods were used to enumerate the number of bacteria and/or phages associated with eyed eggs and in the surrounding environment. The results of the study showed that, with our infection model, it was possible to re-isolate F. psychrophilum associated with eyed eggs after the infection procedure, without affecting the survival of the eggs in the short term. However, this was not possible for F. columnare, as this bacterium grows at higher temperatures than the ones recommended for incubation of rainbow trout eyed eggs. Bacteriophages do not appear to negatively affect the survival of rainbow trout eyed eggs and they do not seem to strongly adhere to the surface of eyed eggs either. Finally, the results demonstrated a strong potential for short term (24 h) phage control of F. psychrophilum. However, further studies are needed to explore if phage control can be maintained for a longer period and to further elucidate the mechanisms of interactions between Flavobacteria and their phages in association with fish eggs.

The fish pathogen Flavobacterium psychrophilum infects farmed salmonids worldwide, and application of bacteriophages has been suggested for controlling disease outbreaks in aquaculture. Successful application of phages requires detailed knowledge about the variability in phage susceptibility of the host communities. In this study, we analysed the genetic diversity of F. psychrophilum hosts and phages from the Baltic Sea area to identify genetic determinants of phage-host interaction patterns. A host range analysis of 103 phages tested against 177 F. psychrophilum strains (18 231 phage–host interactions) identified nine phage clusters, infecting from 10% to 91% of the strain collection. The core genome-based comparison of 35 F. psychrophilum isolates revealed an extremely low overall genomic diversity (>99.5% similarity). However, a small subset of 16 ORFs, including genes involved in the type IX secretion system (T9SS), gliding motility and hypothetical cell-surface related proteins, exhibited a highly elevated genetic diversity. These specific genetic variations were linked to variability in phage infection patterns obtained from experimental studies, indicating that these genes are key determinants of phage susceptibility. These findings provide novel insights on the molecular mechanisms determining phage susceptibility in F. psychrophilum and emphasizes the importance of phages as drivers of core genomic diversity in this pathogen.

The microbial community surrounding fish eyed eggs can harbor pathogenic bacteria. In this study we focused on rainbow trout (Oncorhynchus mykiss) eyed eggs and the potential of bacteriophages against the pathogenic bacteria Flavobacterium psychrophilum and F. columnare. An infection bath method was first established, and the effects of singular phages on fish eggs was assessed (survival of eyed eggs, interaction of phages with eyed eggs). Subsequently, bacteria-challenged eyed eggs were exposed to phages to evaluate their effects in controlling the bacterial population. Culture-based methods were used to enumerate the number of bacteria and/or phages associated with eyed eggs and in the surrounding environment. The results of the study showed that, with our infection model, it was possible to re-isolate F. psychrophilum associated with eyed eggs after the infection procedure, without affecting the survival of the eggs in the short term. However, this was not possible for F. columnare, as this bacterium grows at higher temperatures than the ones recommended for incubation of rainbow trout eyed eggs. Bacteriophages do not appear to negatively affect the survival of rainbow trout eyed eggs and they do not seem to strongly adhere to the surface of eyed eggs either. Finally, the results demonstrated a strong potential for short term (24 h) phage control of F. psychrophilum. However, further studies are needed to explore if phage control can be maintained for a longer period and to further elucidate the mechanisms of interactions between Flavobacteria and their phages in association with fish eggs.

Phage-based approaches have gained increasing interest as sustainable alternative strategies to antibiotic treatment or as prophylactic measures against disease outbreaks in aquaculture. The potential of three methods (oral, bath, and injection) for delivering a two-component phage mixture to rainbow trout fry for controlling Flavobacterium psychrophilum infections and reduce fish mortality was investigated using bacteriophages FpV4 and FPSV-D22. For the oral administration experiment, bacteriophages were applied on feed pellets by spraying (1.6 × 108 PFU g–1) or by irreversible immobilization (8.3 × 107 PFU g–1), using the corona discharge technology (Fixed Phage Ltd.). The fish showed normal growth for every group and no mortality was observed prior to infection as well as in control groups during the infection. Constant detection of phages in the intestine (∼103 PFU mg–1) and more sporadic occurrence in kidney, spleen, and brain was observed. When fish were exposed to F. psychrophilum, no significant effect on fish survival, nor a direct impact on the number of phages in the sampled organs, were detected. Similarly, no significant increase in fish survival was detected when phages were delivered by bath (1st and 2nd bath: ∼106 PFU ml–1; 3rd bath: ∼105 PFU ml–1). However, when phages FpV4 and FPSV-D22 (1.7 × 108 PFU fish–1) were administered by intraperitoneal injection 3 days after the bacterial challenge, the final percent survival observed in the group injected with bacteriophages FpV4 and FPSV-D22 (80.0%) was significantly higher than in the control group (56.7%). The work demonstrates the delivery of phages to fish organs by oral administration, but also suggests that higher phage dosages than the tested ones may be needed on feed pellets to offer fish an adequate protection against F. p